Immunoglobulin A (IgA) nephropathy (IgAN) is one of the most common glomerulopathies worldwide, leading to kidney failure in up to 40% of those affected. This illness is caused by inflammation of the kidney brought about by extensive IgA1 protein deposits in all the renal glomeruli. It is considered likely that any therapy that can safely remove this IgA1 protein from the kidney will reverse the illness. We will develop IgA protease of Haemophilus influenzae as an injectable biological therapeutic for this purpose. Bacterial IgA proteases all have unique substrate specificity for human IgA1. In preliminary studies we have purified Haemophilus IgA protease to homogeneity and developed a mouse model of IgAN in which human IgA1 complexes were directed to the mouse glomeruli. The IgA protease successfully removed these IgA complexes from the kidney in vivo, suggesting the possibility that these enzymes can be used as a biological therapeutic for patients with this disease. The problem we face is that the enzyme is large, MW 109 kDa, and likely to elicit antibodies when injected intravenously. We plan to minimize this immunogenicity with pegylation, epitope masking, and removal of amino acid sequences that are not essential for function. As we reported in the first application, we were successful in preparing crystals of the enzyme that were observed to diffract to ~2.7 E, and this recently led to our success in solving the structure of the Haemophilus influenzae IgA protease to 1.75 E resolution. Because the structure shows the mechanism by which IgA protease recognizes and then cleaves IgA1, we now know how to modify certain regions of the protein without losing bioactivity. Also, the structural analysis suggests several additional ways to reduce antigenicity. In Phase I we will use the recent structure information to introduce two mutational insertions that can be cleaved by furin, a specific enzyme that is intended to reduce the IgA protease size without modifying its function. In Phase I we also will extend our panel of monoclonal antibodies to define where the major epitopes are on the enzyme protein so we can remove one or more these regions to reduce immunogenicity. In Phase II we will make final reductions in enzyme size and will introduce modifications to reduce immunogenicity, and then will compare native and modified forms of the protease for immunogenicity in mice. We will then compare these forms for their effectiveness in removing renal IgA1 in mice, using our validated model of IgAN. Our goal for this fast track project is to produce a modified, effective IgA protease with low antigenicity for treatment of IgAN. The immediate next step after this project is to seek FDA approval to begin clinical trials of our candidate drug. The long term goal is the successful completion of clinical trials, and the entry of our product into the clinical marketplace. PUBLIC HEALTH RELEVANCE: We are developing a new drug that will arrest and reverse Immunoglobulin A (IgA) nephropathy, a kidney disease that begins in children and young adults and often progresses to kidney failure. The illness is caused by material (a protein called IgA) that accumulates in the kidney, and slowly causes inflammation and kidney damage. The drug will be injected into patients, with the intent to remove this protein material. The expectation is that the treatment will arrest kidney damage, and restore kidney function.